On the mechanism of early recovery of specifically depleted lymphoid cell populations by nonspecific activation of T cells.

Specific depletion from normal CBA mouse spleen cells of those bound on pigeon erythrocyte (PRBC) immunoabsorbent columns before transfer of the depleted population into irradiated syngeneic recipients resulted in elimination of the anti-PRBC responsiveness as assessed by rosette (RFC) and hemolytic plaque (PFC) formation. The anti-sheep erythrocyte (SRBC) responses of cell populations treated in the same manner remained unimpaired. When, however, these populations were stimulated with both PRBC and muramyl dipeptide (MDP), an early recovery of specific anti-PRBC responsiveness was produced. PFC response in particular, suddenly increased between the fourth and fifth day after transfer and stimulation thus exhibiting a doubling time of only 4 to 6 hr. This effect of MDP was T-cell dependent since treatment of the depleted population with anti-θ antigen serum and complement hindered early recovery. Depleted populations stimulated with PRBC alone resumed their T-dependent RFC (but not PFC) responsiveness after the eighth day. In spite of the existence of these educated T cells, a second stimulation on the tenth day with PRBC was unable to elicit a specific PFC response. On the other hand stimulation with MDP alone on the day of cell transfer (Day 0) followed by stimulation with PRBC on Day 10 resulted in a specific PFC response on Day 15. Thus, MDP appeared to do more than simply promote education of T cells by antigen. In vitro cultures of depleted populations also recovered their specific reactivity when stimulated by antigen and MDP.     

On the Use of a Simple Physical System Analogy to Study Robustness Features in Animal Sciences

Environmental perturbations can affect the health, welfare, and fitness of animals. Being able to characterize and phenotype adaptive capacity is therefore of growing scientific concern in animal ecology and in animal production sciences. Terms borrowed from physics are commonly used to describe adaptive responses of animals facing an environmental perturbation, but no quantitative characterization of these responses has been made. Modeling the dynamic responses to an acute challenge was used in this study to facilitate the characterization of adaptive capacity and therefore robustness. A simple model based on a spring and damper was developed to simulate the dynamic responses of animals facing an acute challenge. The parameters characterizing the spring and the damper can be interpreted in terms of stiffness and resistance to the change of the system. The model was tested on physiological and behavioral responses of rainbow trout facing an acute confinement challenge. The model has proven to properly fit the different responses measured in this study and to quantitatively describe the different temporal patterns for each statistical individual in the study. It provides therefore a new way to explicitly describe, analyze and compare responses of individuals facing an acute perturbation. This study suggests that such physical models may be usefully applied to characterize robustness in many other biological systems.     

Biological functions of trout pavement-like gill cells in primary culture on solid support: pH(i) regulation, cell volume regulation and xenobiotic biotransformation

This review presents results obtained on rainbow trout gill cells in primary culture on solid support. Ultrastructural analysis showed that cultured gill cells displayed features of pavement cells in situ. Several biological functions have been investigated on these cultured cells. First, it was shown that their intracellular pH at rest and after acidosis is regulated by a Na+/H+ exchanger. Second, gill cells in primary culture can regulate their volume after a cell swelling. Intracellular calcium appears to be involved in this regulation. The effects of different xenobiotics on the capacity of gill cells to regulate their volume are presented. Third, cultured pavement cells contain biotransformation enzymes to metabolize xenobiotics. All these results demonstrate that gill cells in primary culture on solid support represent a promising in vitro model for the study of pavement cells physiology. In conclusion, applications of this culture are discussed and compared with the permeable filter method, together with the limitations and prospects of this in vitro model on solid support.     

Effect of hypotonic shock on cultured pavement cells from freshwater or seawater rainbow trout gills

The effect of hypotonic shock on cultured pavement gill cells from freshwater (FW)- and seawater (SW)-adapted trout was investigated. Exposure to 2/3rd strength Ringer solution produced an increase in cell volume followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also induced a biphasic increase in cytosolic Ca(2+) with an initial peak followed by a sustained plateau. Absence of external Ca(2+) did not modify cell volume under isotonic conditions, but inhibited RVD after hypotonic shock. [Ca(2+)](i) response to hypotonicity was also partially inhibited in Ca-free bathing solutions. Similar results were obtained whether using cultured gill cells prepared from FW or SW fishes. When comparing freshly isolated cells with cultured gill cells, a similar Ca(2+) signalling response to hypotonic shock was observed regardless of the presence or absence of Ca(2+) in the solution. In conclusion, gill pavement cells in primary culture are able to regulate cell volume after a cell swelling and express a RVD response associated with an intracellular calcium increase. A similar response to a hypotonic shock was recorded for cultured gill cells collected from FW and SW trout. Finally, we showed that calcium responses were physiologically relevant as comparable results were observed with freshly isolated cells exposed to hypoosmotic shock.     

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7 beta-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7 beta-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.     

Comparison of the cytotoxic,pro-oxidant and pro-inflammatory characteristics of different oxysterols

Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, refered to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7α-, 7β-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O₂^{⋅ −}) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O₂^{⋅ −} overproduction quantified with hydroethidine was identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, with cholesterol-5α, 6α-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7β-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.     

Quantitative trait loci for magnitude of the plasma cortisol response to confinement in rainbow trout

Better understanding of the mechanisms underlying interindividual variation in stress responses and their links with production traits is a key issue for sustainable animal breeding. In this study, we searched for quantitative trait loci (QTL) controlling the magnitude of the plasma cortisol stress response and compared them to body size traits in five F2 full‐sib families issued from two rainbow trout lines divergently selected for high or low post‐confinement plasma cortisol level. Approximately 1000 F2 individuals were individually tagged and exposed to two successive acute confinement challenges (1 month interval). Post‐stress plasma cortisol concentrations were determined for each fish. A medium density genome scan was carried out (268 markers, overall marker spacing less than 10 cM). QTL detection was performed using qtlmap software, based on an interval mapping method ( http://www.inra.fr/qtlmap ). Overall, QTL of medium individual effects on cortisol responsiveness (<10% of phenotypic variance) were detected on 18 chromosomes, strongly supporting the hypothesis that control of the trait is polygenic. Although a core array of QTL controlled cortisol concentrations at both challenges, several QTL seemed challenge specific, suggesting that responses to the first and to a subsequent exposure to the confinement stressor are distinct traits sharing only part of their genetic control. Chromosomal location of the steroidogenic acute regulatory protein (STAR) makes it a good potential candidate gene for one of the QTL. Finally, comparison of body size traits QTL (weight, length and body conformation) with cortisol‐associated QTL did not support evidence for negative genetic relationships between the two types of traits.